Antiviral compositions consisting of acyclovir and a 2-acetyl pyridine derivative

ABSTRACT

The present invention relates to a novel ribonucleotide reductase inhibitor and new combinations comprising an antiviral compound, such as acyclovir, and a thiocarbonohydrazone ribonucleotide reductase inhibitor for the chemo-therapeutic treatment of virus infections, especially viruses of the herpes group.

This is a continuation of application Ser. No. 07/663,585, filed Mar. 1,1991, U.S. Pat. No. 5,164,395, which is a continuation of Ser. No.07/371,877, filed Jun. 6, 1989 U.S. Pat. No. 5,021,437.

FIELD OF INVENTION

The present invention relates to new antiviral combinations for thechemotherapeutic treatment of virus infections, especially viruses ofthe herpes group, and to certain compounds for use in such combinations.

BACKGROUND INFORMATION

During the last fifteen years, various antiviral chemotherapeutic agentshave been developed for clinical evaluation. A problem with thedevelopment of such agents is that, unlike bacteria, viruses are notfree-living organisms and are dependent for replication on the lifeprocesses of the host cell which they are infecting. It is thereforehighly desirable for the antiviral agent to exert its effectspecifically on the replicative processes of the virus rather than onthe corresponding processes of normal (non-infected) cells. Theantiviral agents so far developed act via a variety of mechanisms toexert their antiviral effects. These mechanisms involve inhibition ofdifferent stages in the process of viral replication in the host cells.

One particular stage of replication at which the virus is susceptible toinhibition is the stage of nucleic acid replication, i.e., theproduction of DNA from DNA, RNA from RNA, or DNA from RNA (depending onwhether the virus is a DNA or an RNA virus), where the viral DNA or RNAacts as a template for the production of new DNA or RNA. In the case ofDNA viruses, the production of new viral DNA involves the interaction ofthe enzyme DNA polymerase with the constituent nucleotides (specifiallydeoxyribonucleotides) which act as building blocks for the new DNA.Antiviral action at this stage generally involves the use of"fraudulent" or deleterious nucleotides which mimic the normal viralmaterials and either compete for DNA polymerase and/or are incorporatedinto the viral DNA chain to make it non-functional.

These "fraudulent" or deleterious nucleotides comprise a triphosphatederived from a nucleoside analog which is converted by enzymes firstinto the monophosphate and then subsequently into the diphosphate andfinally into the triphosphate. An example of this type of antiviralagent is the marketed compound, acyclovir, i.e.,9-(2-hydroxyethoxymethyl)guanine (U.S. Pat. No. 4,199,574), whichcontains an acyclic side-chain in the 9-position of guanine comparedwith a cyclic sugar residue in this position in guanosine. The antiviralmechanism of action of acyclovir is believed to involve first itsconversion to acyclovir monophosphate by the enzyme thymidine kinase,which is specific to herpes-infected cells. Once formed, acyclovirmonophosphate is conversed by normal cellular enzymes (kinases) via thediphosphate to acyclovir triphosphate (ACV-TP). Acyclovir triphosphateis believed to serve as an inhibitor of viral DNA polymerase since itresembles the natural nucleotide substrate, deoxyguanosine triphosphate(dGTP), and as a result competes with dGTP for binding to the DNApolymerase and thus competitively inhibits the effectiveness of theenzyme and consequently viral replication. When ACV-TP acts as asubstrate for DNA polymerase it becomes incorporated into the viral DNAchain, but since it lacks the 3'-hydroxyl group present on the cyclicsugar moiety of the natural nucleotide substrate, it presumably acts asa DNA chain terminator. It also apparently inactivates the viral DNApolymerase. Thus, viral replication is prevented.

The antiviral effect of acyclovir, and related compounds which operatevia an analogous mode of action, is believed to involve competitiveinhibition, apparent inactivation of the viral DNA polymerase, andtermination of the growing DNA chain.

A disadvantageous aspect of a competitive inhibitor is that the normalsubstrate may accumulate and become more effective in competitivelyblocking the binding of the inhibitor. In this manner, the build up of,for example, dGTP may hinder the binding of ACV-TP to the polymerase andthereby prevent subsequent inhibition and termination of viral DNAprocessing.

European Patent Specification 135,713 (U.S. Pat. No. 4,758,572)discloses antiviral combinations of nucleoside analogs andthiosemicarbazone ribonucleotide reductase inhibitors. U.S. Pat. No.4,719,221 discloses the thiosemicarbazones in European PatentSpecification 135,713.

SUMMARY OF THE INVENTION

It has now been discovered that a certain ribonucleotide reductase (RR)inhibitor, as disclosed herein, has anti-herpes virus activity.Furthermore, this RR inhibitor significantly potentiates the efficacy ofantiviral compounds, such as acyclovir. The combination of RR inhibitorand antiviral compound has the effect, in virus-infected cells, ofdecreasing the pool of deoxynucleotide substrate of virus DNA polymeraseand, surprisingly, increasing the triphosphate form of the antiviralcompound. Thus, the ratio of triphosphate form of the antiviral compoundto the competing deoxynucleotide substrate of viral DNA polymerase, suchas deoxyguanosine triphosphate, is greatly improved, and the binding ofthe triphosphate form of the antiviral compound to the viral DNApolymerase is facilitated.

The net result is that the use of a certain RR inhibitor, as disclosedherein, in combination with an antiviral compound of the above describedtype results in a surprising synergistic increase in antiviral efficacyin comparison with the individual antiviral effects of the components ofthe combination. The RR inhibitor of this invention is more potent insynergy with acyclovir in vivo and substantially less toxic than thethiosemicarbazone RR inhibitors cited in the above-mentioned EuropeanPatent Specification.

OBJECT OF THE INVENTION

According to a feature of the present invention, there is provided acombination of (1) an antiviral compound which is capable of beingconverted in vivo, via a metabolic pathway, which includes a stepdependent upon virus-induced enzyme, to an inhibitor of, or analternative substrate for, viral DNA polymerase, and (2) aribonucleotide reductase inhibitor which is a compound of formula (I),namely 2-acetylpyridine 5-[(2-chloroanilino)thiocarbonyl]thiocarbonohydrazone. ##STR1##

The present invention also provides the above-defined combination foruse in medical therapy e.g. the treatment of viral diseases in the humanand animal body. Examples of such viral diseases include those caused byviruses of the herpes family, for example, herpes simplex 1 and 2viruses (HSV 1, HSV 2), varicella zoster virus (VZV), cytomegalovirus(CMV) and Epstein-Barr virus (EBV), but other herpes virus infectionscan also be treated (e.g. feline herpes virus infections).

The invention further provides a method for the treatment of viraldiseases in a human or animal body which comprises administering to thehuman or animal body an effective non-toxic amount of a combination asdefined above. It will be appreciated that, in accordance with thepresent invention, the antiviral compound and the RR inhibitor may beadministered simultaneously (in the same or different pharmaceuticalformulations) or sequentially. In the latter case, however, thecomponents of the combination are preferably administered within asufficiently short interval to ensure that a synergistic antiviraleffect is achieved.

The invention further provides use of a combination according to thepresent invention in the manufacture of a medicament for use in thetreatment or prophylaxis of a virus infection, particularly herpes,selected from HSV 1, HSV 2, VZV, CMV and EBV.

The present invention also provides:

a) A method of therapeutically increasing the pool size of a deleterioussubstrate and/or an inhibitor of viral DNA polymerase in a mammal havinga viral infection and receiving an antiviral compound, which depends onviral-induced enzyme(s) for conversion to said deleterious substrateand/or inhibitor in the mammal, and the improvement of administering tosaid mammal an RR inhibitor of formula (I) or a pharmaceuticallyacceptable salt thereof in an amount effective for increasing the poolof said inhibitor and/or deleterious substrate of viral DNA polymerasein said mammal; and

b) A method of potentiating in a mammal having a viral infection theantiviral activity of an antiviral compound being administered to saidmammal, which depends on viral-induced enzyme(s) for conversion to adeleterious substrate and/or inhibitor of viral DNA polymerase, andwhich comprises administering to said mammal an effective potentiatingamount of an RR inhibitor of formula (I) or a physiologically acceptablesalt thereof.

An advantage of the combination according to the invention is that itenables one to obtain an improved antiviral efficacy. Furthermore, alower dosage of an antiviral compound (compared with the compound usedalone) can be efficacious, thereby improving the therapeutic index ofthe antiviral compound. Thus, for example, the combination may be usedto treat conditions which would otherwise require relatively largedosages of the antiviral compound at which toxicity problems may occur.

The combination according to the invention is especially applicable tothe treatment of herpes simplex types 1 and 2 infections and varicellazoster virus infections (e.g., shingles).

DESCRIPTION OF THE PREFERRED EMBODIMENT

With regard to the antiviral compound, this can be selected from anycompound that is activated in vivo by virus-induced enzyme(s) as definedabove. Such compounds are generally substrates for an appropriate kinaseenzyme of viral origin which phosphorylates the compounds to form amonophosphate which, per se or in a metabolically transformed form, isthen phosphorylated (by kinase enzymes of either viral or cellularorigin) to form the diphosphate and finally the triphosphate DNApolymerase inhibitor or deleterious substrate. The use of an antiviralcompound that is selectively phosphorylated by viral enzymes rather thanby cellular enzymes provides a greater concentration of thephosphorylated (activated) antiviral compound in infected cells than innon-infected cells. Thus the activated antiviral compound causesselective toxicity to the virus, inhibiting virus replication. It ispreferred to use an antiviral compound that is not only a DNA polymeraseinhibitor but is also, when incorporated into the viral DNA chain, achain terminator and, possibly, an apparent inactivator of the viral DNApolymerase.

Thus, for example, acyclovir, as mentioned above, is converted byvirus-coded thymidine kinase (but not to any substantial extent bycellular thymidine kinase) to the monophosphate which is then convertedto the diphosphate and triphosphate via cellular enzymes. Acyclovirtriphosphate is also a DNA chain terminator. The mechanism of acyclovirand other antiviral compounds is described by E. de Clerq in "New Trendsin Antiviral Chemotherapy", Archives Internationale de Physiologie et deBiochimie, 87(2), 353-395 (1979) and by P. Furman et al. in "Acyclovir:Mechanism of Action", Human Herpes Infections, edited by C. Lopez and B.Roizman, pp. 129-138, Raven Press, New York (1986).

The antiviral compound employed in the combinations according to theinvention may be selected for example from acyclovir and analogsthereof, e.g., those compounds of formula (II) ##STR2## (wherein X isoxygen or sulfur, R is hydrogen, hydroxy or amino and Y is hydrogen orhydroxymethyl) and pharmaceutically acceptable salts and esters thereof.

In addition to acyclovir, examples of preferred compounds of formula(II) for use in the present invention include:

9-[(2-hydroxy-1-hydroxymethylethoxy)methyl]guanine (see U.S. Pat. No.4,609,662) as well as prodrugs that are converted in vivo into the abovecompounds, e.g. 2-amino-9-(2-hydroxyethoxymethyl)adenine and9-[(2-hydroxy-1-hydroxymethylethoxy)methyl]-2,6-diaminopurine. See U.S.Pat. No. 4,323,573 and G.B. Patent 2,104,070.

Alternatively, the antiviral compound may be1-[2-hydroxy-1-(hydroxymethyl) ethoxymethyl]cytosine or pharmaceuticallyacceptable salts and esters thereof. See European Patent Specification167,385.

The above-mentioned nucleoside analogs also include the pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to a human subject, is capable of providing(directly or indirectly) the antivirally active metabolite or residuethereof. See European Patent Specification 272,065.

The above-defined RR inhibitor of formula (I) and its pharmaceuticallyacceptable salts are novel and represent a further feature of thepresent invention, since they have been found to potentiate, in asynergistic manner, the antiviral effects of the antiviral compoundsdescribed above. The RR inhibitor of formula (I) also has activityagainst herpes viruses which renders the compound useful per se in thetreatment of herpes virus infections.

Salts of the RR inhibitor, according to the invention, which may beconveniently used in therapy, include pharmaceutically acceptable basesalts, derived from an appropriate base, such as alkali metal (e.g.sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and NX⁺ 4(wherein X is C₁₋₄ alkyl) salts. Suitable pharmaceutically acceptablesalts also include, but are not limited to, those prepared from thefollowing acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, salicylic, p-toluene-sulfonic, tartaric, citric,acetic, methanesulfonic, formic, succinic, naphthalene-2-sulfonic,isethionic, lactobionic and benzenesulfonic. The pharmaceuticallyacceptable salts of the above RR inhibitors may be prepared inconventional manner for example by treatment with the appropriate baseor acid.

The combinations according to the invention may be administered to thesubject concerned in conventional manner. As indicated above, theantiviral compound and the RR inhibitor may be administeredsimultaneously (e.g. in a unitary pharmaceutical formulation) orseparately (e.g. in separate pharmaceutical formulations). In general,the combinations may be administered by the topical, oral, rectal orparenteral (e.g. intravenous, subcutaneous or intramuscular) route. Thedosage of the combination will depend on the condition being treated,the particular antiviral compound and RR inhibitor concerned, and otherclinical factors such as the weight and condition of the patient and theroutes of administration of the compounds. However, for administrationby the oral route, a dosage of the antiviral compound of 1 to 150mg/kg/day, more preferably 5-125 mg/kg/day, and most preferably 15 to 80mg/kg/day, is generally sufficient. The amount of RR inhibitor in thecombination will be determined from the amount of antiviral compoundspecified above and the desired ratio of antiviral compound to RRinhibitor.

The ratio of antiviral compound to RR inhibitor is preferably in therange of about 0.5:1 to 50:1 (w/w), more preferably 1:1 to 30:1 (w/w),and most preferably 5:3 (w/w). Thus when 25 mg/kg/day of acyclovir isadministered to a subject, 15 mg/kg/day of 2-acetylpyridine5-[(2-chloroanilino)thiocarbonyl]thiocarbonohydrazone would also beadministered.

For convenience, the antiviral compound and RR inhibitor are preferablyadministered in a unitary pharmaceutical formulation. Thus, the presentinvention further provides a pharmaceutical formulation comprising acombination according to the invention, together with at least onepharmaceutical carrier or excipient, the antiviral compound and RRinhibitor being present in the formulation in a ratio whereby asynergistic antiviral effect is achieved.

Formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal, parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal andepidural) administration. The formulation may conveniently be presentedin unit dosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional compatible pharmaceutically-activematerials which enhance antiviral effectiveness or facilitatecombination therapy (such as potentiating agents, antivirals,antimicrobials, antipruritics, astringents, local anesthetics oranti-inflammatory agents), or may contain materials useful in physicallyformulating various dosage forms of the composition of presentinvention, such as excipients, dyes, perfumes, fragrances,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. These materials, when added, should not unduly interferewith the penetration enhancement of the compositions of this invention.Such formula modifications to improve cosmetic acceptability are wellwithin the skill of workers in the cosmetic and dermatological arts and,by themselves, constitute no part of the present invention.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing predetermined amounts of the active ingredients; aspowders or granules; as solutions or suspensions in an aqueous liquid ora non-aqueous liquid; or as oil-in-water emulsions or water-in-oilliquid emulsions. A tablet may be made by compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine, the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein.

For infections of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the combined active ingredients in a total amount of,for example, 0.075 to 30% w/w, preferably 0.2 to 20% w/w and mostpreferably 0.4 to 15% w/w. When formulated in an ointment, the activeingredients may be employed with either paraffinic or a water-miscibleointment base.

Alternatively, the active ingredients may be formulated in a cream withan oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, for exampleat least 30% w/w of a polyhydric alcohol, i.e., an alcohol having two ormore hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof. The topical formulation may desirably include a compound whichenhances absorption or penetration of the active ingredient through theskin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier, it desirably comprises a mixture of at least oneemulsifier with a fat or an oil or with both a fat and an oil.Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat.

Together, the emulsifier(s) with or without stabilizer(s) make-up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulsifiers and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, Myrj andBirj.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus, the cream should preferably bea non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredients are dissolved or suspended insuitable carrier, especially an aqueous solvent for the activeingredients. The antiviral active ingredients are preferably present insuch formulations in a concentration of 0.5 to 20%, advantageously 0.5to 10% and particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert bases such as gelatin and glycerin, or sucroseand acacia; and mouth washes comprising the active ingredients in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for nasal administration wherein the carrier is asolid include a coarse powder having a particle size for example in therange 20 to 500 microns which is administered in a manner in which snuffis taken, i.e., by rapid inhalation through the nasal passage from acontainer of the powder held close up to the nose. Suitable formulationswherein the carrier is a liquid, for administration as for example anasal spray or as nasal drops, include aqueous or oil solutions of theactive ingredients.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredients such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient. Suitable formulations alsoinclude aqueous and non-aqueous sterile suspensions which may containsuspending agents and thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solution and suspensions may be prepared from sterile powders,granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of the activeingredients.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude flavoring agents.

Antiviral Activity

The ribonucleotide reductase inhibitor of the present invention wasshown by use of the dye uptake method described by McLaren, Ellis andHunter in Antivir. Res. 3,223-234 (1983) to potentiate the in vitroantiviral effects of acyclovir. Vero cells (20,000 cells/well) wereincubated in microtiter wells for 24 hours. Various concentrations ofacyclovir and the ribonucleotide reductase inhibitor were added and thecells were infected with 30 infectious particles/well of HSV-1 or HSV-2.After 3 days incubation, the antiviral effects were assessed bydetermining the prevention of the cytopathic effects of the viruses.Examples of the synergistic antiherpes activity of 2-acetylpyridine5-[(2-chloroanilino)thiocarbonyl]thiocarbonhydrazone (compound B); andacyclovir (ACV) are given below.

An example of the synergistic antiherpes activity of Compound B and ACV:

    ______________________________________                                                   IC.sub.50.sup.a                                                                                      Fold                                        Compound(s)  Observed  Calculated.sup.b                                                                         potentiated.sup.c                           ______________________________________                                        ACV          1.6       1.6        --                                          ACV plus 0.4 μM                                                                         0.84      1.6        1.9                                         Compound B (0%).sup.d                                                         ACV plus 0.6 μM                                                                         0.62      1.6        2.5                                         Compound B (0%).sup.d                                                         ACV plus 0.9 μM                                                                         0.33      1.6        4.8                                         Compound B (0%).sup.d                                                         ACV plus 1.3 μM                                                                         0.19      1.6        8.4                                         Compound B (0%).sup.d                                                         ACV plus 2.0 μM                                                                         0.15      1.1        7.3                                         Compound B (11%).sup.d                                                        ______________________________________                                         .sup.a Concentration (μM) required to inhibit HSC2 replication by 50%.     .sup.b Theoretical value for the combination of ACV and Compound B.           .sup.c Ratio of calculated IC.sub.50 to observed IC.sub.50.                   .sup.d Parenthetical number is the % inhibition of virus replication by       Compound B alone.                                                        

The following Examples are provided by way of illustration of thepresent invention and should in no way be construed as limitationthereof.

EXAMPLES RR Inhibitors

Preparation 1: 2-Acetylpyridine thiocarbonohydrazone

To a solution of distilled 2-acetylpyridine (Aldrich Chemical Co.,Milwaukee, Wis. 53233), 33.3 g, in 300 mL of methanol was addedthiocarbohydrazide (Sigma Chemical Co., St. Louis, Mo., 63178), 29.18 g.The resulting mixture was heated at reflux, under nitrogen, overnight. Aprecipitate was collected by filtration, washed with cold methanol, anddried to give 56.0 g (97%) of 2-acetylpyridine thiocarbonohydrazone as awhite crystalline solid, m.p. 185°-186° C. (dec.).

¹ H-NMR: (DMSO-d₆) δ2.34 (s, 3H, CH₃), 5.00 (br s, 2H, NH₂), 7.36 (ddd;1H; J=1.0, 4.9, 7.5; aromatic CH), 7.77 (dt; 1H; J=1.7, 7.8; aromaticCH), 8.53 (m, 2H, aromatic CH), 9.96 (br s, 1H, NH), 10.31 (s, 1H, NH).

Preparation 2: 2-Acetylpyridine5-[(methylamino)thiocarbonyl]thiocarbonohydrazone

A 1 L 3-neck flask fitted with an overhead mechanical stirrer,condenser, glass stopper, and a nitrogen line was charged with2-acetylpyridine thiocarbonohydrazone, 5.37 g, and 500 mL of absoluteethanol. The resulting mixture was heated at reflux for 5 min todissolve most of the solid material. The mixture was allowed to coolbelow reflux temperature and methyl isothiocyanate (Aldrich), 2.20 mL(2.35 g), was added. The reaction mixture was heated at reflux for 30min, allowed to cool to room temperature, and subsequently cooled in anice-water bath. A precipitate was collected by filtration, washed withcold ethanol, and dried in vacuo to give 6.74 g (93.3%) of2-acetylpyridine 5-[(methylamino)thiocarbonyl]thiocarbonohydrazone as awhite crystalline solid, m.p. 178.0°-178.5° C. (dec.).

¹ H-NMR: (DMSO-d₆) δ2.39 (s, 3H, COCH₃), 2.86 (d, 3H, J=4, NCH₃), 7.38(m, 1H, aromatic CH), 7.85 (m, 1H, NH), 8.56 (m, 2H, aromatic CH), 9.43(br s, 1H, NH), 10.32 (br s, 1H, NH), 10.82 (br s, 1H, NH).

The following compound (Example 1) was prepared using the general methoddescribed in Preparation 2:

Novel RR Inhibitor Example 1

2-Acetypyridine 5-[(2-chloroanilino)thiocarbon]1]thiocarbonohydrazone

Reaction of 2-acetylpyridine thiocarbonohydrazone, 7.00 g, and2-chlorophenyl isothiocyanate (Trans World, Chemicals, Inc., ChevyChase, Md. 20815), 5.67 g, in 245 mL of dry DMF gave 8.74 g of a tanpowder. Recrystallization from DMF/H₂ O afforded 5.69 g (45%) of thetitle compound as a light yellow powder, m.p. 159°-160° C. (dec.).

¹ H-NMR: (DMSO-d₆) δ2.48 (s, 3H, CH₃), 7.17-8.58 (m, 8H, aromatic CH),9.39 (br s, 1H, NH), 9.95 (br s, 1H, NH), 10.52 (br s, 1H, NH), 10.96(s, 1H, NH).

Elemental Analysis: Calcd. for C₁₅ H₁₅ N₆ ClS₂. 0.25 H₂ O (mw 383.41,parent mw 378.91): C, 46.99; H, 4.07; N, 21.92; Cl, 9.25; S, 16.72.Found: C, 46.90; H, 4.07; N, 21.88; Cl , 9.25; s, 16.67.

Pharmaceutical Formulations

In the following Examples, the antiviral compound is acyclovir and theRR inhibitor is 2-acetylpyridine5-[(2,-chloroanilino)thiocarbonyl]thiocarbonohydrazone.

    ______________________________________                                        Tablet             Amount                                                     ______________________________________                                        RR Inhibitor       300 mg                                                     Antiviral Compound 500 mg                                                     Lactose            105 mg                                                     Starch              50 mg                                                     Polyvinylpyrrolidinone                                                                            20 mg                                                     Magnesium Stearate  10 mg                                                                        985 mg                                                     ______________________________________                                    

The active compounds are mixed with the lactose and starch and are wetgranulated with a solution of the polyvinylpyrrolidinone. The granulesare dried, sifted, and blended with magnesium stearate and compressed.

    ______________________________________                                        Capsule            Amount                                                     ______________________________________                                        RR Inhibitor       300 mg                                                     Antiviral Compound 500 mg                                                     Lactose            100 mg                                                     Sodium Starch Glycollate                                                                          10 mg                                                     Polyvinylpyrrolidinone                                                                            10 mg                                                     Magnesium Stearate  3 mg                                                                         923 mg                                                     ______________________________________                                    

The active compounds are mixed with the lactose and sodium starchglycollate and are wet granulated with a solution of thepolyvinylpyrrolidinone. The granules are dried, sifted, and blended withthe magnesium stearate and filled into hard gelatin capsules.

    ______________________________________                                        Cream (1)         Amount                                                      ______________________________________                                        RR Inhibitor      6.00 g                                                      Antiviral Compound                                                                              10.00 g                                                     Glycerol          2.00 g                                                      Cetostearyl Alcohol                                                                             6.75 g                                                      Sodium Lauryl Sulfate                                                                           0.75 g                                                      White Soft Paraffin                                                                             12.50 g                                                     Liquid Paraffin   5.00 g                                                      Chlorocresol      0.10 g                                                      Purified Water to 100.00 g                                                    ______________________________________                                    

The active compounds are mixed with purified water and glycerol andheated to 70° C. The remaining ingredients are heated together at 70° C.The two parts are added together and emulsified. The cream is cooled andfilled into containers.

    ______________________________________                                        Cream (2)         Amount                                                      ______________________________________                                        RR Inhibitor       6.00 g                                                     Antiviral Compound                                                                              10.00 g                                                     Mineral Oil, Heavy                                                                               5.00 g                                                     Polawax            7.50 g                                                     Propylene Glycol  40.00 g                                                     White Petrolatum  12.50 g                                                     Sodium Edetate     0.10 g                                                     Purified Water to 100.00 g                                                    ______________________________________                                    

A solution of sodium edetate in water is heated to 70°-75° C. and addedto a mixture of the mineral oil, white petrolatum and polawax, heated tothe same temperature. The propylene glycol is added and the mixture isallowed to cool to 50°-55° C. The two active compounds are added, mixedand allowed to cool to 25°-30° C. Sufficient purified water is added toachieve the proper batch weight. The cream is filled into containers.

    ______________________________________                                        Intravenous Injection                                                                             Amount                                                    ______________________________________                                        RR Inhibitor        300 mg                                                    Antiviral Compound  500 mg                                                    Glycerol            200 mg                                                    Sodium Hydroxide solution qs                                                                      pH 7.0-7.5                                                Water for Injections to                                                                            10 mL                                                    ______________________________________                                    

The active compounds and the mannitol are dissolved in a part of thewater for injections. The pH is adjusted with the sodium hydroxidesolution and the solution is made up to volume with additional water forinjections. Under aseptic conditions, the solution is sterilized byfiltration and filled into sterile vials. Water is removed byfreeze-drying. The vials are sealed under an atmosphere of nitrogen andare closed with a sterile closure and a metal collar.

Toxicology

Rats were dosed orally with 2-acetylpyridine 5-((2-chloroanilino)thiocarbonyl)thiocarbonohydrazone, 60 mg/kg 1 day, for 30 days. At 15and 30 days, the hematological profile of the compound showed less than10% deviation from control values for all the parameters measured. Therats also had normal colored urine and normal body weight.

I claim:
 1. An anti-herpetic composition comprising an amount of a firstcompound acyclovir and a second compound 2-acetylpyridine-5-thiocarbonohydrazone, the first and second compounds being present in an amount toprovide an anti-herpetic synergistic effect, and the ratio of the firstcompound to the second compound being 0.5:1 to 50:1 (w/w).
 2. Thecomposition of claim 1 wherein the ratio of the first compound to thesecond compound is 1:1 to 30:1 (w/w).
 3. The composition of claim 1wherein the ratio of the first compound to the second compound is 5:3(w/w).
 4. An anti-herpetic composition comprising an amount of a firstcompound acyclovir and a second compound which is a pharmaceuticallyacceptable salt of2-acetylpyridine-5-[(2-chloroanilino)thiocarbonyl]thiocarbonohydrazone,the first and second compounds being present in an amount to provide ananti-herpetic synergistic effect, and the ratio of the first compound tothe second compound is 0.5:1 to 50:1 (w/w).
 5. The composition of claim4 wherein the ratio of the first compound to the second compound is .5:1to 30:1 (w/w).
 6. The composition of claim 4 wherein the ratio of thefirst compound to the second compound is 5:3 (w/w).